Genomic Stability and Long-term Transgene Expression in Poplar

Stable expression of foreign genes over the entire life span of a plant is important for long-lived organisms such as trees. For transgenic forest trees, very little information is available on long-term transgene expression and genomic stability. Independent transgenic lines obtained directly after transformation are initially screened in respect to T-DNA integration and transgene expression. However, very little consideration has been given to long-term transgene stability in long-lived forest trees. We have investigated possible genome wide changes following T-DNA integration as well as long-term stability of transgene expression in different transgenic lines of hybrid aspen (Populus tremula × Populus tremuloides) that are up to 19 years old. For studies on possible genome wide changes following T-DNA integration, four different independent rolC-transgenic lines were subjected to an extensive AFLP study and compared to the non-transgenic control line. Only minor genomic changes following T-DNA integration could be detected. To study long-term transgene expression, six different independent rolC-transgenic lines produced in 1993 and since that time have been kept continuously under in vitro conditions. In addition, 18 transgenic plants belonging to eight independent rolC-transgenic lines transferred to glasshouse between 1994 and 2004 were chosen to determine the presence and expression of the rolC gene. In all transgenic lines examined, the rolC gene could successfully be amplified by PCR tests. Both, the 19 years old tissue cultures and the up to 18 years old glasshouse-grown trees revealed expression of the rolC transgene, as demonstrated by the rolC-phenotype and/or northern blot experiments confirming long-term transgene expression.